Prostate cancer (PC) is the most diagnosed tumor in males and ranks as the second leading cause of male mortality in the western world. The CD39 and CD73 enzymes play a crucial role in cancer regulation by degrading nucleotides and forming nucleosides. This study aimed to investigate the expression of the CD39 and CD73 enzymes as potential therapeutic targets for PC. The initial part of this study retrospectively analyzed tissue samples from 23 PC patients. Using the TissueFAXS^TM cytometry platform, we found significantly higher levels of CD39—labeling its intensity compared to CD73. Additionally, we observed a correlation between the Gleason score and the intensity of CD39 expression. In the prospective arm, blood samples were collected from 25 patients at the time of diagnosis and after six months of treatment to determine the expression of CD39 and CD73 in the serum extracellular vesicles (EVs) and to analyze nucleotide hydrolysis. Notably, the expression of CD39 in the EVs was significantly increased compared to the CD73 and/or combined CD39/CD73 expression levels at initial collection. Furthermore, our results demonstrated positive correlations between ADP hydrolysis and the transurethral resection and Gleason score. Understanding the role of ectonucleotidases is crucial for identifying new biomarkers in PC.